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B Galvan

Publications and source records attributed to B Galvan.

5 recordsLinked to original sources
PubMed1997

Quantitative reverse transcriptase-polymerase chain reaction for prostate-specific antigen mRNA.

OBJECTIVE: To develop a quantitative reverse transcriptase-polymerase chain reaction assay for monitoring the prostate-specific antigen (PSA) mRNA. METHODS: PSA mRNA is amplified, in parallel, with the mRNA of beta-actin, a housekeeping gene. The ratio of the amplification products obtained reflects the relative amount of PSA mRNA with respect to actin mRNA. During PCR, digoxigenin-dUTP is incorporated in the amplified sequences. The PCR products are analyzed separately by time-resolved immunofluorometric hybridization assays, using specific probes immobilized in microtiter wells. The hybrids are reacted with alkaline phosphatase-labeled anti-digoxigenin antibody. The phosphate ester of fluorosalicylate is used as a substrate. The fluorosalicylate produced forms a fluorescent complex with Tb(3+)-EDTA which is measured by time-resolved fluorometry. RESULTS: The hybridization assays for both PSA and actin amplification products show linearity in the range of 1.4-110 pmol/L. The exponential phase of PCR amplification extends up to 200,000 and 100,000 PSA and actin cDNA molecules, respectively. We prepared mixtures containing various numbers of LNCaP cells in one million cells that do not express PSA and used them as samples in the proposed assay. The ratio of the fluorescence values obtained after analysis of PSA and actin amplification products is linearly related to the number of LNCaP cells in the range of 20 to 3000 cells. Reproducibility studies demonstrate %CVs for the fluorescence ratios of 14.7, 11.8, and 12.2 when samples containing 150, 300 and 1600 LNCaP cells were analyzed (n = 4). CONCLUSIONS: A quantitative analytical methodology is provided for monitoring PSA mRNA. The assay is expected to be beneficial in the study of prostate cancer spread.

Humans↗
PubMed1996

Bioluminescence hybridization assays using recombinant aequorin. Application to the detection of prostate-specific antigen mRNA.

We developed microtiter well-based bioluminescence hybridization assays using the photoprotein aequorin as a reporter molecule. The target DNA was hybridized simultaneously with a capture probe and a detection probe. The capture probe was immobilized on the wells through digoxigenin/anti-digoxigenin interaction. The detection probe was biotinylated. The hybrids were determined by using aequorin covalently attached to streptavidin or complexes of biotinylated aequorin with streptavidin. The luminescence was then measured in the presence of excess Ca2+. The optimized protocols showed linearity in the range from 5 amol to 10 fmol of target DNA. In combination with reverse transcriptase polymerase chain reaction, the proposed assay was applied to the detection of the mRNA for prostate-specific antigen (PSA). PSA mRNA from a single cell, in the presence of one million cells that do not express PSA, was detected with a signal-to-background ratio of 2.5. Typical CVs obtained were 6%.

Aequorin↗
PubMed1996

Fluorometric and time-resolved immunofluorometric assays for protein-tyrosine phosphatase activity.

OBJECTIVE: To develop sensitive nonisotopic assays for protein-tyrosine phosphatase (PTP) activity. METHODS: The fluorometric assay is based on the fact that phosphotyrosine but not tyrosine forms highly fluorescent complexes with Tb3+. Thus, PTP activity can be followed by measuring the decrease of fluorescence due to hydrolysis of phosphotyrosine. The time-resolved immunofluorometric assay employs tyrosine-phosphorylated substrates, immobilized on microtitre wells. After incubation with PTP, the remaining phosphotyrosine residues are reacted with an antiphosphotyrosine antibody. The immunocomplexes formed are detected with an alkaline phosphatase (ALP)-labeled second antibody. The phosphate ester of 5' fluorosalicylate (FSAP) is used as substrate. The fluorosalicylate produced forms highly fluorescent complexes with Tb3+ - EDTA in alkaline solution. The fluorescence is measured with a time-resolved fluorometer. RESULTS: The truncated form of the T-cell protein tyrosine phosphatase (TCdeltaC11 PTP) was determined in the range 1100-36,500 U/L by the fluorometric assay and 36-7100 U/L by the time-resolved immunofluorometric assay. CONCLUSIONS: The two nonisotopic assays should prove beneficial for the determination and study of various PTP.

Evaluation Studies as Topic↗
PubMed1995

Detection of prostate-specific antigen mRNA by reverse transcription polymerase chain reaction and time-resolved fluorometry.

We have developed a time-resolved fluorometric hybridization assay for detecting prostate-specific antigen (PSA) mRNA amplified by reverse transcription polymerase chain reaction. During PCR, digoxigenin-11-dUTP is incorporated into the amplified product. An oligonucleotide internal to the primers is used as a specific probe, being biotinylated and captured on streptavidin-coated microtiter wells. Denatured PCR product hybridizes with the probe, and the hybrids are detected with an alkaline phosphatase-labeled antidigoxigenin antibody. We used the phosphate ester of fluorosalicylic acid as the substrate. The fluorosalicylate produced forms a highly fluorescent ternary complex with Tb(3+)-EDTA, which we can measure by time-resolved fluorometry. A signal-to-background ratio of 10 was obtained when 160 PSA cDNA molecules were present in the preamplification sample. Also, mRNA corresponding to one LNCaP cell in the presence of 10(6) PSA-negative cells can be detected (signal-to-background ratio of 3.1). Samples containing 100, 1000, and 50,000 LNCaP cells gave CVs of 12.4%, 4.9%, and 6.8%, respectively (n = 10).

Fluorometry↗